Periodontitis aggravates COPD through the activation of γδ T cell and M2 macrophage

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a chronic systemic inflammatory disease with high morbidity and mortality. Periodontitis exacerbates COPD progression; however, the immune mechanisms by which periodontitis affects COPD remain unclear. Here, by constructing periodontitis and COPD mouse models, we demonstrated that periodontitis and COPD could mutually aggravate disease progression. For the first time, we found that the progression was associated with the activation of γδ T cells and M2 macrophages, and M2 polarization of macrophages was affected by γδ T cells activation. In the lung tissues of COPD with periodontitis, the activation of γδ T cells finally led to the increase of IL 17 and IFN γ expression and M2 macrophage polarization. Furthermore, we found that the periodontitis-associated bacteria Porphyromonas gingivalis (P. gingivalis) promoted the activation of γδ T cells and M2 macrophages ex vivo. The data from clinical bronchoalveolar lavage fluid (BALF) samples were consistent with the in vivo and ex vivo experiments. For the first time, our results identified the crucial role of γδ T-M2 immune mechanism in mediating periodontitis-promoted COPD progression. Therefore, targeting at periodontitis treatment and the γδ T-M2 immune mechanism might provide a new practical strategy for COPD prevention or control. IMPORTANCE Periodontitis exacerbates chronic obstructive pulmonary disease (COPD) progression. For the first time, the current study identified that the impact of periodontitis on COPD progression was associated with the activation of γδ T cells and M2 macrophages and that M2 polarization of macrophages was affected by γδ T cells activation. The results indicated that targeting at periodontitis treatment and the γδ T-M2 immune mechanism might provide a new practical strategy for COPD prevention or control.

has been identified as the keystone bacteria in the dysbiosis of oral microbiota and periodontitis occurrence and development (10)(11)(12).
Oral health status seriously impacts respiratory diseases via several aspects (13)(14)(15): (i) Changes in oral health status leads to oral immune imbalance, after which local oral immune cells migrate to the lung, causing an immune imbalance of lung tissue and, thus, affect lung diseases; (ii) Changes in oral health status can also promote the progression of lung disease by affecting systemic inflammation.(iii) Oral health status is closely related to changes of oral microbiota, and then oral microorganisms affect the lung flora through inhalation and colonization on lung tissue, leading to lung immune imbalance and inflammatory response, and affecting the progression of lung diseases.At present, the lung microbiota changes and immune imbalance caused by ectopic colonization of oral microorganisms in the lungs is one of the main ways that oral health status affects respiratory diseases.Studies have shown that P. gingivalis in the mouth of periodontitis patients or experimental mice can transfer and colonize on the lungs, causing lung inflammation and injury (16)(17)(18).Periodontal pathogens such as P. gingivalis, Fusobac terium.nucleatum, Prevotella oralis, and Aggregatibacter actinomycetemcomitans have been identified in the lung aspiration of pneumonia patients (2).Bronchial microbial studies of asthma indicated the presence of four specific bacterial groups, two of which, Fusobacterium and Porphyromonas, are known periodontal pathogenic bacteria (19).Similarly, higher diversity and rates of microbial colonization were found in the lungs of patients with COPD, and microorganisms from the mouth and upper respiratory tract were detected (2,20).Previous studies have indicated that the oral periodontitisassociated bacteria P. gingivalis acted as one of the main causes of the life-threatening infectious disease aspiration pneumonia and that P. gingivalis gingipains could lead to the upregulation of inflammatory factors, including TNF, IL-6, IL 17, and C-reactive protein (21).In short, it indicated that there was a microbial link between periodontitis and COPD state.
Dozens of studies have confirmed that there is, indeed, a positive correlation between periodontitis and COPD (22,23).People with severe periodontitis have a much higher relative risk of COPD compared with periodontal healthy counterparts (24).The frequency of lung function attenuation and exacerbation in COPD patients with periodontitis is more serious than in COPD patients with good periodontal health (25).Through basic periodontal treatment and oral health maintenance, the frequency of lung function attenuation and exacerbation in COPD patients can be well controlled (26,27).However, the specific mechanism by which periodontitis affects COPD progression still requires further exploration.γδ T cells are T cells that perform innate immune functions, which could secret IL 17 and initiate inflammatory responses (28).Studies have shown that oral microorganisms can promote the proliferation of in situ γδ T cells in the lungs and secretion of IL 17 to mediate lung inflammation and disease status (29,30).However, the effect of γδ T cells on COPD is controversial and needs further study.Under the stimulation of IL 4/IL 10/IL 13, macrophages could become M2-polarized, after which cytokines such as MMP9 and MMP12 are produced by M2-polarized macrophages to promote the process of parenchymal injury (31,32).The occurrence of COPD is related to the enhancement of immune response mediated by macrophage polarization (33,34).The correlation between M2 polarization and COPD is controversial, and the mecha nism of M2 polarization in periodontitis-promoted COPD remains unclear and needs further study.Through chemokines, cytokines, and cell surface receptors, γδ T cells and macrophages may undergo some inter-regulation (35)(36)(37).IL 17 and IFN γ produced by γδ T cells can play an inflammatory role; meanwhile, IL 17 can also promote the M2 polarization of lung macrophages (38,39).
Based on the above background, we hypothesized that in the periodontitis state, the major periodontal pathogenic bacteria, as represented by P. gingivalis, may transfer and colonize on the lung tissue, causing microbiota changes in the lung, further leading to pulmonary immune imbalance and promoting the accumulation and proliferation of γδ T cells in the lung.On the one hand, γδ T cells produce IL 17 and IFN γ to directly promote the COPD process; on the other hand, the production of IL 17 by γδ T cells further promotes the M2 polarization of alveolar macrophages and contributes to COPD progression.Therefore, this study intends to determine whether periodontitis promotes COPD progression by regulating γδ T cells and M2 macrophages through a series of experiments, including in vivo animal models and ex vivo bacterial cell co-culture, and clinical sample detection, in order to provide new directions for clinical COPD patient management and disease control from the perspective of oral health.

COPD and periodontitis could mutually promote disease progression
To observe the effect of periodontitis on COPD progression, we constructed four groups of animal models: B group, blank control group (healthy mice); P group: periodontitis group (mice with periodontitis only); C group, COPD group (mice with COPD only); CP group, COPD with periodontitis group (mice with both COPD and periodontitis).As shown in Fig. 1, periodontitis and COPD could mutually promote disease progression.Periodontitis aggravates the lung function decrease of COPD, with declining forced expiratory volume in 0.05 second (FEV0.05)and ratio of forced expiratory volume in 0.05 second to forced vital capacity (FEV0.05/FVC)(Fig. 1A).H&E observation demonstra ted the most seriously dilated and fractured alveolar wall, narrowest airway and most neutrophils infiltration in COPD with periodontitis group (CP group) (Fig. 1B).Similarly, the severity of periodontitis was also most pronounced in the CP group, with the highest amount of alveolar bone resorption (Fig. 1C and D).

Microbiota homeostasis changed under the COPD and P. gingivalis-periodon titis state
During the periodontitis model construction procedure, mice were orally infected with P. gingivalis (1 × 10 9 CFU/mL, 0.2 mL/mice) every other day.To analyze whether P. gingivalis could migrate to and colonize on the lungs, we assessed the abundance of P. gingivalis in the lung tissue and found that periodontitis promoted the increase of P. gingivalis abundance in COPD lung tissue (Fig. 2A).To further explore whether P. gingivalisperiodontitis caused changes in the microbiota of lung tissue, we carried out 16S rRNAsequencing analysis.The α diversity indices, β diversity index and differences in the relative abundances of bacterial taxa were compared among the groups to evaluate the microbiota change of lung tissue under different disease states.Through 16S rRNAsequencing analysis, we observed that the microbiota homeostasis of lung tissue underwent an observable change.Compared to the blank control group (B group), the α diversities, including Shannon, Simpson, and observed species diversity, of the periodon titis (P), COPD (C), and COPD with periodontitis (CP) groups were decreased (Fig. 2B through D); meanwhile, β diversity analysis showed that the P, C, and CP groups showed a different distribution in comparison with the B group (Fig. 2E).Moreover, composition analysis also demonstrated some differences (Fig. 2F through H).Compared to B group (20.25% ± 2.632%), Proteobacteria was the most abundant phylum in the P (63.03% ± 18.57%), C (70.47% ± 7.595%), and CP (81.47% ± 5.152%) groups, and in the CP group, the periodontitis condition could further increase the abundance of Proteobacteria compared to C group (Fig. 2F).Similarly, at the class level, Betaproteobacteria was the most abundant taxon in the P (60.95% ± 17.2%), C (68.71% ± 7.085%), and CP (81.23% ± 5.96%) groups compared to the B group (16.08% ± 2.326%), and periodontitis could further increase the abundance of Betaproteobacteria in the COPD lung (Fig. 2G).At the genus level, compared to B group (34.53% ± 15.36%, 7.157% ± 4.387%, respectively), Ralstonia and Pelomonas were the two most abundant taxa in the P (52.42% ± 15.42%, 25% ± 11.63%, respectively), C (64.51% ± 12.46%, 20.92% ± 5.721%, respectively), and CP (72.15% ± 3.359%, 26.28% ± 4.887%, respectively) groups, and both of these taxa were increased in the CP group compared to C group (Fig. 2H).Interestingly, it was observed that in the CP group, Proteobacteria (40) and Ralstonia (41), which are associated with lung inflammation, were increased in comparison to the C group.Some oral-associated pathogenic bacteria, including Lactobacillus, Prevotella, and Bactriodes, were also detected in the lung tissues.Compared to C group, some periodontal inflammatoryassociated bacteria, including Bacteroidetes, Bacteroides, and Prevotella, showed increased relative abundance in CP group (Table S1).In short, P. gingivalis-periodontitis affected the microbiota homeostasis of lung tissue.

In the development of periodontitis promoting COPD, γδ T cells and polariza tion of macrophages were activated
To further explore the potential immune mechanism by which periodontitis affected COPD, we analyzed the gene expressions of immune cytokines.In the early stage of COPD (2 weeks of smoke exposure), RT-qPCR results showed that the expression levels of Il 17 and Ifn γ, M1 polarization, and M2 polarization-related markers and inflammatory cytokines (CD86, iNOS, Il 1β, Il 23, Mmp 9, Mmp 12, Il 4) in COPD lung tissues were significantly up-regulated under the condition of periodontitis (Fig. 3A; Fig. S1a).ELISA results showed that periodontitis significantly up-regulated the levels of IL 17 and IFN γ in COPD lung tissues (Fig. 3B) and serums (Fig. S1b).These data preliminarily indicated that IL 17 and IFN γ production and macrophage polarization might play important roles in mediating periodontitis-promoting COPD.Considering that γδ T cells possess the ability to produce both IL 17 and IFN γ, we hypothesized that periodontitis might affect COPD progression by promoting the activation of γδ T cells and macrophage polarization.Interestingly, both flow cytometry analysis and immunofluorescence (IF) observation suggested that periodontitis promoted the aggregation and expansion of γδ T cells in COPD lung tissue and, simultaneously, macrophage polarization in CP group lung tissues was significantly increased, including M1 and M2 polarization (Fig. 3C and  D; Fig. S1c).These data preliminarily suggested that periodontitis could promote COPD progression through the activation of γδ T cells and polarization of macrophages.

The promotive effect of periodontitis on COPD progression through γδ T cells and M2 macrophages was prolonged over time
Further extending smoke exposure time by another 2 weeks (4 weeks of smoke exposure, totally), in the later stages of COPD, we found that the mutually reinforcing interaction between periodontitis and COPD was more pronounced in the mouse models with the extended smoke exposure time (Fig. 3E and F).In CP group, lung function was further decreased, with further decreases in FEV0.05 value and FEV0.05/FVC (Fig. 3E).Lung tissue lesions were more obvious; airway inflammatory infiltration was aggravated (Fig. 3F).Similarly, alveolar bone resorption was also further aggravated (Fig. S3a).
Gene expression analysis showed that periodontitis still up-regulated the gene expression of Il 17, IFN γ, and macrophage polarization-related markers and inflammatory cytokines in COPD lung tissue in the later stage (Fig. S2a).ELISA analysis also confirmed that periodontitis up-regulated the levels of IL 17 and IFN γ in COPD lung tissue and serum and that the up-regulation effects were significantly stronger than those of the early stage (Fig. S2b and c).
Flow cytometry analysis and IF observation also showed that periodontitis further promoted the aggregation and proliferation of γδ T cells and the polarization of macrophages in lung tissue in the CP group (Fig. 3G and H; Fig. S3b).Among them, periodontitis significantly increased the γδ T cells and M2 macrophage polarization in COPD lung tissue compared to the earlier stage model, while the M1 polarization did not change significantly (Fig. 3G).Therefore, we speculated that periodontitis played an important role in promoting the progress of COPD mainly through the up-regulated γδ T activation and M2 polarization, and in the following experiments, we focused on the role of γδ T activation and M2 polarization.Further analysis showed that periodontitis could further up-regulate the ability of γδ T cells to express IL 17 and IFN γ in lung tissue during the later stage of COPD (Fig. 3I).These results indicated that the promotive effect of periodontitis on COPD progression was prolonged over time and activation of IL 17 + γδ T cells, IFN γ + γδ T cells, and M2 macrophages played important roles in how periodontitis affected COPD progression.The periodontitis-associated bacteria P.

gingivalis could activate γδ T cells and M2 macrophages
To evaluate whether the periodontitis-associated bacteria P. gingivalis had the potential to activate γδ T cells and M2 macrophages, we carried out a series of ex vivo experiments.These ex vivo experiments were conducted using mice materials, including mice lung tissue lymphocytes, BALF, and peripheral blood mononuclear cells (PBMC).As shown in Fig. 4; Fig. S4, P. gingivalis could stimulate the γδ T cells activation in lung lymphocytes and promote their ability to produce IL 17 and IFN γ (Fig. 4A; Fig. S4a).In addition, P. gingivalis could also stimulate the M1 and M2 polarization of macrophages in lung BALF (Fig. 4B; Fig. S4a through S4c).Furthermore, in the PBMC environment, co-coculture with P. gingivalis elevated γδ T cells, IL 17 + γδ T cells, and IFN γ + γδ T cells (Fig. 4C; Fig. S4b), M1 polarized macrophages and M2 polarized macrophages (Fig. 4D; Fig. S4b through S4d), as well as the levels of IL 17 and IFN γ levels in the supernatants (Fig. 4E).To further confirm the important role of γδ T cells, we carried out γδ T cells inhibition treatment.Under the inhibitor-treated PBMC environment (the γδ-TCR monoclonal antibodies were first intraperitoneally injected in vivo, and 24 h later, the peripheral blood of mice was collected to obtain the inhibitor-treated PBMC), we observed that the levels of IL 17 and IFN γ in the supernatants were significantly decreased (Fig. 4F);also, the activation potentials of P. gingivalis on γδ T cells and M2 macrophage polarization were significantly decreased (Fig. 4G).While, under γδ-TCR monoclonal antibody treatment, the M1 polarization of macrophages was not significantly affected, regardless of the presence or absence of P. gingivalis stimulation (Fig. S5).These results indicated that P. gingivalis could activate γδ T cells activation and M2 macrophage polarization and that the M2 macrophage polarization was affected by the γδ T cells.Therefore, combined with the above experimental results, we hypothesized that periodontitis could aggravate COPD through the γδ T-M2 immune mechanism.On the one hand, periodontitis promoted γδ T cells aggregation and proliferation and the production of IL 17 and IFN γ in lung tissue, which could aggravate COPD progres sion directly; on the other hand, the activated γδ T cells mediated M2 polarization of macrophages (IL 17 + γδ T cells in the lung tissue possessed the potential to induce M2 polarization of macrophages), M2 macrophages could further aggravate COPD progression.

Inhibition of γδ T cells affected the promotion of COPD by periodontitis
To further confirm the essential effect of γδ T activation in periodontitis' influence on COPD progression and M2 polarization in vivo, we constructed mouse models with γδ-TCR monoclonal antibody inhibition treatment.As shown in Fig. 5, in the anti-γδ T groups (C-Anti group and CP-Anti group), lung functions and lung lesions were significantly improved compared to those of the C and CP groups, which phenotypically confirmed the important role of γδ T cells in how periodontitis affects COPD (Fig. 5A  and B).However, alveolar bone resorption was not significantly affected by the γδ-TCR monoclonal antibody inhibition treatment (Fig. S6).
Flow cytometry and immunofluorescence observation verified the significant reduction of γδ T cells expansion in the lung tissues in the anti-γδ T groups (C-Anti group and CP-Anti group) compared to the groups without γδ-TCR monoclonal antibody treatment (C group and CP group, respectively); meanwhile, compared to C group and CP group, COPD severity in C-Anti group and CP-Anti group was significantly improved, respectively.These results indicated that γδ-TCR monoclonal antibody treatment was ± STD. *P < 0.05, ns, not significant.Lung-lym, the lymphocytes of lung tissue; Lung-lym +P.g, the lymphocytes of lung tissue cocultured with P. gingivalis; BALF, bronchoalveolar lavage fluid; BALF +P.g, bronchoalveolar lavage fluid cocultured with P. gingivalis; PBMC, peripheral blood mononuclear cells; PBMC +P.g, peripheral blood mononuclear cells cocultured with P. gingivalis; PBMC-Anti, PBMC group with γδ-TCR monoclonal antibody treatment; P. g + PBMC-Anti, P. g + PBMC group with γδ-TCR monoclonal antibody treatment.associated with milder COPD symptoms.Interestingly, inhibition of the γδ T cells also resulted in a decrease of M2 macrophages in the lung tissue (Fig. 5C, G and H).Consistent with the ex vivo results, under γδ-TCR monoclonal antibody treatment, the M1 polariza tion of macrophages in the lungs was not significantly affected, regardless of the presence or absence of periodontitis (Fig. S7).These results again demonstrated that the activation of γδ T cells in lung tissue partly induced the M2 polarization of macrophages and promoted disease progression in the periodontitis-promoted COPD model (Fig. 5C through G).Similarly, the ELISA and RT-qPCR results indicated that γδ-TCR monoclonal antibody treatment decreased the levels of IL 17 and IFN γ in lung tissue (Fig. S8a) and serum (Fig. S8b), as well as gene expression levels in lung tissue (Fig. S8c and d).

Research
Briefly, these data suggested that activation of γδ T cells played a crucial role in both M2 macrophage polarization and COPD progression.M2 macrophages acted as another immune-based cause of COPD progression and were affected by γδ T cells in this study.Therefore, it could be concluded that in the current periodontitis-promoted COPD model, activation of γδ T cells affected M2 macrophage polarization and periodontitis exacerbated COPD through the promotion of the γδ T-M2 immune mechanism.

Enhanced IFN γ and IL 17 levels and M2 polarized gene expressions in clinical COPD with periodontal pathogenic bacteria BALF samples
The data presented above demonstrated that periodontitis aggravated COPD progres sion through activation of γδ T cells and M2 macrophages.The expression of IL 17 and IFN γ by activated γδ T cells directly promoted COPD, and, simultaneously, the expressed IL 17 induced M2 polarization of macrophages, which also promoted COPD.To further confirm the important roles of IFN γ and IL 17 levels and M2-polarized gene expressions in clinical COPD with periodontal pathogenic bacteria BALF samples, 53 clinical BALF samples were collected.After P. gingivalis detection analysis, 25 samples that were positive for the presence of P. gingivalis were assigned to the COPD-P.g group, and the other 28 samples that lacked P. gingivalis were assigned to the control group (COPD-no P.g).The characteristics of clinical samples in the two groups were shown in Table S2.The systemic conditions of the patients in the two groups were consistent, and there were no significant differences in age, gender, lifestyle habits such as smoking, systemic disease conditions such as hypertension, diabetes, etc.We further reviewed the COPD disease status of the sampled patients, and the results showed that among the 25 subjects with P. gingivalis detection, 20 samples were diagnosed as being in the acute exacerbation stage, while among the 28 samples in the control group, 19 were in the acute exacerba tion stage of COPD.That was to say, the incidence of acute exacerbation of COPD in the experimental group (80.00%) was higher than that in the control group (67.86%), which could reflect to a certain extent that the existence of periodontal pathogenic bacteria was related to the exacerbation of COPD.ELISA measurements suggested that higher levels of IL 17 and IFN γ in the BALF supernatants of COPD with periodontal pathogenic bacteria (P.gingivalis) (Fig. 6A).Gene expression analysis also indicated that the expression levels of IL 17/IFN γ and M2 related genes ARG1, CD206, MMP9, MMP12, TGF β, IL 4, and IL10 were higher in COPD BALF samples with the presence of P. gingivalis (Fig. 6B).Our results demonstrated that in the clinical environment, IFN γ and IL 17 levels and M2 polarized genes might be important factors in COPD with periodontitis.

DISCUSSION
Periodontitis is a chronic inflammatory disease of the periodontal tissue caused by plaque biofilm, which acts as a potential risk factor for a variety of systemic diseases, including cardiovascular disease, diabetes, hypertension, various cancers, COPD, etc. (5,42,43).As is commonly known, globally, chronic respiratory diseases act as the leading cause of death and morbidity, among which COPD causes the most deaths, and is considered the third leading cause of death worldwide (44).COPD is a devastating lung disease characterized by an incomplete and reversible airflow restriction that is usually progressive and associated with the lungs' abnormal inflammatory response to harmful particles or gases (45).Many previously published studies reported the positive association between periodontitis and COPD (22,23), but the potential immune mechanisms were unclear.
In the current study, we found that periodontitis could aggravate COPD progression (Fig. 1), which was consistent with previous studies (1,46).Further microbe analysis indicated that microbiota composition changed under disease state, and that periodonti tis caused an increase of lung inflammation associated bacteria, such as Proteobacteria and Ralstonia.To some degree, this phenomenon indicated that periodontitis could promote lung inflammation development.Like previous studies (2,19,20), we also observed the microorganism communication between lung tissue and the oral cavity.Many oral bacteria were detected in the lung tissues, including Lactobacillus, Prevotella, Bactriodes, and P. gingivalis.Periodontitis also caused the increase of two main bacterial taxa, Ralstonia and Pelomonas, in the COPD lungs.Moreover, some oral periodontal-asso ciated pathogenic bacteria, including Prevotella, Bactriodes, and P. gingivalis were also detected with increased relative abundance in the COPD with periodontitis lung tissue compared to those in the COPD group.Therefore, it could be concluded that periodonti tis affected the microbiota homeostasis of the lung.
γδ T cells perform innate immune functions and act as an important subset of T cells, which play an important role in inflammatory immunity via the secretion of cytokines, including IFN γ and IL 17 (28).IL 17 is a pivotal cytokine that regulates lung immunity and inflammation and has shown the capacity to mediate COPD progression (47,48).IFN γ is another crucial cytokine that is associated with COPD.Numerous studies demonstrated that the IFN γ levels as well as IFN γ-producing cells were increased in COPD patients and experimental mouse models, while effective COPD treatment resulted in decreased IFN γ levels (49)(50)(51).Moreover, a recent study indicated that Th17/Th1 cells capable of secreting both IL 17 and IFN γ were more pathogenic in COPD (52).
In lung tissue, γδ T cells constitute a major tissue-resident T cell component and play important roles in maintaining the homeostasis of lung tissue (53,54).The effects of γδ T cells have two sides in lung tissue.Some studies have indicated that γδ T cells helped to activate host defense against bacterial, viral, and fungal infections and participated in maintaining lung tissue homeostasis (53,54).Under inflammation state of lung tissue, γδ T cells displayed a tissue reparative effect through the production of IL 17 and were found to be associated with inflammation control (54,55).Additionally, γδ T cells in lung tissue also showed protective effects towards cystic fibrosis and lung cancers (53,56,57).However, on the contrary, numerous other studies have indicated that γδ T cells might act with a crucial pathogenic effect in lung diseases.γδ T cells and their IL 17 expression are required for pulmonary inflammation and injury caused by ozone exposure (30).IL 17A-producing γδ T and Th17 lymphocytes mediated lung inflammation and acute lung injury in experimental silicosis (58).Jin et al. (29) implicated that commensal-bacteria activated γδ T cells in lung tissue and promoted lung adenocarcinoma through IL 17 production.Data in Vigeland's study elucidate the critical role of IL 17A + γδ T cells in promoting chronic inflammation and fibrosis of the lung tissue (39).
Similarly, there has been some controversy about the role of γδ T cells in COPD progression.Pons et al. (59) reported blunted γδ T-lymphocyte response in the COPD disease state.Similarly, Urboniene et al. ( 60) investigated γδ T cells in induced sputum (IS) and bronchoalveolar lavage (BAL) and found reduced amount of γδ T cells in IS and BALF from COPD patients compared those from asthmatic or healthy subjects, which indicated that γδ T cells might have a protective effect in preventing COPD.However, inconsistent with this, Majo et al. observed in clinical lung tissue samples that γδ T cells significantly increased in the lungs of smoker and emphysema patients (61).Smoking causes or exacerbates COPD by promoting γδ T proliferation and IL 17 production (48).Serum amyloid A promotes lung neutrophil accumulation and COPD by increasing γδ T cells and the associated expression of IL 17 (62).In this study, we found that γδ T cells played an important role in the cigarette smoke exposure-induced COPD disease model, suggesting that smoking led to the expansion of γδ T cells in lung tissue and contributed to the occurrence of COPD (Fig. 3 and 5), consistent with Majo's and Bozinovski's results (48,61).More importantly, we further found that γδ T cells were important targets for mediating the promotive effect of periodontitis on COPD.In the presence of periodonti tis, the severity of COPD disease was much greater, the proportion of γδ T/IL17 + γδ T/IFN γ + γδ T cells in COPD lung tissue was significantly up-regulated, and the contents of IL 17 and IFN γ were also significantly increased (Fig. 3; Fig. S1 and S2).However, when γδ T cells were inhibited, the proportion of γδ T/IL 17 + γδ T/IFN γ + γδ T cells in lung tissue was significantly reduced, the contents of IL 17 and IFN γ were decreased, the severity of COPD disease was significantly decreased, and the promotive effect of periodontitis on COPD was also significantly affected (Fig. 5; Fig. S8).Therefore, these results suggested that periodontitis contributed to COPD progression by promoting γδ T cells activation in COPD lung tissue.
Macrophages are innate immune cells and play important roles in homeostasis and host defense.The development of COPD is associated with enhanced immune response mediated by macrophage polarization (33,34,63,64).A majority of alveolar macro phages are normally non-polarized in healthy lung tissue, but in lung tissue of COPD, there is a significant increase of macrophage polarization and co-expression of M1 and M2 macrophages (65,66).Moreover, both M1-polarized and M2-polarized macrophages in the lung tissue increased significantly with the progression of smoke and COPD severity (65).However, the role of distinct macrophage phenotypes (M1 versus M2) in COPD is unclear and which macrophage phenotype (M1 versus M2) is more predomi nant in COPD progression is still controversial.Through literature review, Lee et al. (64) concluded that M1-associated marker (iNOS) and cytokines (IL-1β, IL-6, IL-8, TNF-α) were increased in COPD patients, indicating the pathogenic effect of M1 polarization in COPD.LncRNA MIR155HG promoted COPD by upregulating M1 polarization and downregulat ing M2 polarization of lung macrophages (67).Similarly, Sun's data indicated ergosterol showed therapeutic effect toward COPD by decreasing M1 polarization and increasing M2 polarization (68).While, dozens of studies have pointed out that M2 polarization of macrophages was the essential pathogenesis of COPD.Eapen et al. (69) observed that in COPD patients, luminal macrophages showed a dominant polarization of M2 phenotype and BALF showed increased cytokines of the M2 profile, including CCL22, IL-4, IL-13, and IL-10.Liu et al. (70) emphasized the important role of M2 polarization of macrophage and suggested that through suppressing M2 macrophage polarization, effective components combination (ECC) could improve airway remodeling of COPD.In the current study, we found that periodontitis promoted macrophage polarization in COPD lung tissue, including M1 polarization and M2 polarization, and the effect of periodontitis on M2 polarization in COPD lung tissue was significantly enhanced with the disease duration, which is consistent with the study of Feng et al. (71), while, M1 polarization did not change significantly with the disease duration.Therefore, we assumed that in the later stage of the disease, M2 polarization played an important role in mediating the promotive effect of periodontitis on COPD.
Generally, IL 4, IL10, and IL 13 are considered the more common markers for inducing M2 polarization; however, existing studies have shown that in the lung tissue, γδ T cells could regulate M2 polarization of macrophages by producing IL17 and that M2 polarization could be significantly inhibited if γδ T cells was deficient or IL17 was blocked.In lung tissue, IL 17 expressed by activated γδ T cells could induce macrophage proliferation and M2 polarization, thus further affect disease progression (30,38,39).This procedure had been demonstrated in both pulmonary inflammation and lung fibrosis (38,39) as well as in OSCC with periodontitis models (35).Therefore, does such an immune regulatory mechanism also exist in the COPD disease model and can periodon titis further promote COPD progression by regulating this mechanism?In the current study, we found that M2 polarization was influenced by the activation of γδ T cells in lung tissues in COPD with periodontitis.The increase of γδ T and IL17 + γδ T cells could further induce the increase of M2 polarization (Fig. 4 and 5).Similarly, under the condition of γδ T cells inhibition treatment, γδ T and IL17 + γδ T cells were significantly reduced, and M2 polarization was also significantly decreased (Fig. 4 and 5).While, under γδ-TCR monoclonal antibody treatment, M1 polarization of macrophages in the lungs was not significantly affected, regardless of the presence or absence of periodontitis (Fig. S7).These results again demonstrated that the activation of γδ T cells in the lung tissue could partly induce the M2 polarization of macrophages and promote disease progression in the periodontitis-promoted COPD model.Importantly, in clinical BALF samples, we obtained similar results.In COPD BALF samples containing the periodontal pathogenic bacteria P. gingivalis, the levels of IL 17 and IFN γ and the gene expressions of IL 17, IFN γ, and M2 polarization associated genes, including ARG1, CD206, MMP9, MMP12, TGF β, IL 4, and IL10, were significantly upregulated (Fig. 6), indicating the important roles of IL 17 and IFN γ and M2 macrophages in periodontitis-aggravated COPD in the clinical samples.While, in the disease models of current research, the regulatory mechanism of M1 polarization may have a more complex regulatory network, which needs to be further explored in the future.In the current disease model, periodontitis promoted the activation of γδ T cells in the lung.On the one hand, IFN γ and IL17 produced by activated γδ T cells directly promoted the progression of COPD.On the other hand, IL17 produced by activated γδ T cells further promoted M2 polarization, thereby aggravating the progression of COPD.However, in this disease model, M1 polarization might be regulated by a variety of inflammatory and immune factors, not just IFN γ.And in our future studies, we will further explore the regulatory network of M1 polarization in this disease model.Moreover, a novel concept mentioned that some novel markers (CD38, Gpr18, and Fpr2 for M1 polarization, Egr2 and c-Myc for M2 polarization) (72) were recommended to be contained in the macrophage polarization-associated studies, and in the future's experimental study, we will further contain these markers.
Through the in vivo, ex vivo, and clinical samples analysis, we could conclude that in COPD with periodontitis, the microbiota homeostasis of lung tissue changed, and at the same time, an immune imbalance of lung tissue occurred.Periodontitis promoted the activation of γδ T cells, leading to γδ T cells, IL17 + γδ T cells, and IFN γ + γδ T cells expansion in the COPD lung tissue.On the one hand, IL 17 and IFN γ directly promoted the progression of COPD, and, simultaneously, IL 17 further induced M2 polarization of macrophages, thereby contributing to COPD progression (Fig. 7).
However, there are some limitations in this research.The first one is the short duration of the later-stage model.In this study, the later-stage model (4-week model) only extended the cigarette smoke exposure time by 2 weeks compared to the earlier-stage model (2-week model), which might be related to the fact that in this research, the decrease of the lung functions and the increase of some immune cells (γδ T cells and M2 macrophages) were modest.If the cigarette smoke exposure could be extended for a longer period of time, these changes in the later-stage model might be more pro nounced.In our upcoming research, we will further explore the changes of disease severity and investigate the increases of γδ T cells and M2 macrophages with the animal models of many other longer disease duration-time points.The second limitation is the imbalance between the groups in the proportion of patients in acute exacerbation stage.If the acute exacerbation status of the sampled patients between the two groups was the same, it could be better emphasis the role of periodontal pathogenic bacteria in upregulating the inflammatory gene expression and inflammatory cytokines content in the lung.And in our forthcoming study, we will further overcome the difficulty of sample collection and obtain the samples without such limitation, to more accurately reflect the difference between the detection of periodontal pathogenic bacteria and the patient's lung function status.
In summary, we observed that periodontitis could exacerbate COPD progression and identified for the first time that the γδ T-M2 immune mechanism played an important role in mediating periodontitis promoting COPD.The presence of periodontitis intensi fied the activation of γδ T cells and M2 macrophages in COPD lung tissue, resulting in the aggravation of COPD severity.Therefore, targeting at periodontitis treatment and the γδ T-M2 immune mechanism might be a new practical strategy for the prevention or control of COPD.

Experimental animal model
Mouse models were constructed to study the promotion ability and possible immune mechanism that periodontitis affected COPD.The animal model experiment was approved by the animal research committee of West China School of Stomatology, Sichuan University (WCHSIRB-D-2020-127). Male 7-week-old-specific pathogen free (SPF) C57BL/6J mice purchased from Dashuo Biological Technology (Chengdu, China) were randomly divided into several groups (5 mice/group).For periodontitis construction, the 5-0 silk ligatures were tied around the maxillary second molars of mice.Following, mice were infected with P. gingivalis (1 × 10 9 CFU/mL, 0.2 mL/mice) every other day.Briefly, every other day, at about eight o 'clock in the morning, freshly prepared 1 × 10 9 CFU/mL P. gingivalis (0.2 mL/mice) were centrifuged and resuspended with 2% carboxymethylcel lulose (CMC) solution.And then, the CMC-resuspended P. gingivalis were dipped and transferred onto the disposable oral-specific micro applicators.Finally, mice were orally infected with P. gingivalis by brushing the free gingival area slightly using the bacterial containing disposable oral-specific micro applicators.One week later, COPD models were started to construct using newly purchased cigarettes (Marlboro, 12 mg tar/1.0mg nicotine; Philip Morris, Richmond, VA) and porcine pancreatic elastase (PPE, RHAWN catalog no.R028727).Mice were exposed to mainstream cigarette smoke (CS) for 2 h every day through the Cigarette Smoke Generators TSE system (TSE Systems China).Two days before CS exposure was begun and on the second day of CS exposure, mice under anesthesia underwent an intratracheal injection of phosphate-buffered saline (PBS) dissolved PPE (2.5 U/mouse each time, in total, 5 U/mouse).For the early-stage COPD model, mice were exposed to CS for 2 weeks, and for the late-stage COPD model, the CS exposure time was extended for an additional 2 weeks.For the anti-γδ T groups (6 mice/group for B group and P group; 9 mice/group for the other four COPD-associated groups), the mice were intraperitoneally injected with γδ-TCR monoclonal antibody (200 ug/mouse; BioXCell catalog no.BE0070) every 2-3 days.

Lung function test
Lung function was measured with the EMMS eSpira Forced Maneuvers system according to the manufacture's instruction.FEV0.05 value and FEV0.05/FVC value were used to compare the lung function of each group.

H&E staining and methylene blue staining
Five-micrometer paraffin-embedded lung tissue sections were prepared for hematoxylin and eosin (H&E) staining with the H&E staining kit (Solarbio catalog no.G1120) to observe the morphological changes and to analyze disease severity.
The maxillae were stained with 1% methylene blue (Solarbio catalog no.G1303) as described by Wei et al. (35) and imaged with a microscope.The severity of bone resorption, represented by the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC), was measured to analyze periodontitis severity.

16S rRNA-sequencing
Freshly collected lung tissues were quickly frozen in liquid nitrogen and then transported on dry ice to the Personal Biotechnology company (Shanghai Personal Biotechnology Co., Ltd., China) for 16S RNA-sequencing (V3-V4) on the Illumina platform (Novaseq 6000).The data analysis was carried out with QIIME2 (2019.4).

Enzyme-linked immunosorbent assay (ELISA)
IL 17 (BioLegend catalog no.436204) and IFN γ (BioLegend catalog no.430804) ELISA kits were used to measure the levels of IL 17 and IFN γ in mouse serum samples, lung tissues samples, and the cell culture supernatant samples, respectively.

RT-qPCR
For gene expression levels analysis, total RNA of each lung tissue sample was extracted with RNA extraction kit (Yeasen catalog no.19221ES50) and reversely transcribed with RNA reverse kit (Yeasen catalog no.11141ES60).Then, the cDNA samples were used for gene expression level analysis (Gapdh was used to normalize the expressions of different genes) with the RT-qPCR kit (Syber Green, Yeasen catalog no.11201ES08).The expression levels were calculated with the 2 −△△CT method (75).The specific primers for the tested genes were designed using PrimerBanK (https://pga.mgh.harvard.edu/primerbank/) and synthesized by TSINGKE Biological Technology company (Sichuan, Chengdu), the sequences are listed in Table S3.
For the detection of P. gingivalis abundance in lung tissue, the gDNA obtained above underwent RT-qPCR procedure.P. gingivalis abundances were calculated with the 2 −△CT method (76) and displayed with fold changes to the blank control group.

Ex vivo experiment
The lymphocytes of mouse lung tissues were extracted with the Percoll Cell separation fluid kit (Biosharp catalog no.BS909).BALF were collected with 1 mL sterile PBS and centrifuged to collect the cell precipitate.Lymphocytes or BALF cell precipitate were co-cultured with P. gingivalis in DMEM containing 10% FBS for 24 h.
The peripheral blood mononuclear cells (PBMC) of mice blood were extracted with Peripheral blood lymphocyte separation solution (Solarbio catalog no.P6340).PBMC were co-cultured with P. gingivalis for 24 h.For the anti-γδ T treatment ex vivo part, γδ-TCR monoclonal antibodies (200 ng/mL; BioXCell catalog no.BE0070) were added.Briefly, for the anti-γδ T treatment groups, the inhibitor was first intraperitoneally injected in vivo, and 24 h later, the peripheral blood of mice was collected to obtain the inhibitor-treated PBMC, and then, the obtained PBMC was cultured in vitro.For the corresponding control groups (without γδ-TCR monoclonal antibody treatment), an equal amount of PBS was intraperitoneally injected in vivo, 24 h later, the peripheral blood of mice was collected to obtain PBMC without inhibitor treatment, and then, the obtained PBMC was cultured in vitro.
Flow cytometry procedures were performed and analyzed as described above.

FIG 2 PFIG 3
FIG 2 P. gingivalis-periodontitis affected homeostasis of the lung microbiota.(a) The relative abundance of P. gingivalis in lung tissues was determined by RT-qPCR.(b-d) α-diversity of the lung tissue was analyzed for each group and presented with Shannon, Simpson, and observed species indices.(e) β diversity of the lung tissue in each group was presented with PCOA index.(f-h) Lung microbial community composition was analyzed.The percentages of the major phyla, classes, and genera according to 16S rRNA sequencing were shown.*P < 0.05, ns, not significant; B, blank control; P, periodontitis; C, COPD; CP, COPD with periodontitis.

FIG 3 ( 7 FIG 4
FIG 3 (Continued) γδ; green color, CD206 or CD86.Following, we further extended the smoke exposure time to 4 weeks and analyzed the effect of periodontitis on COPD in the time-extended (late-stage) model.(e) In the late stage of COPD, the lung function test results of each group and comparison with the corresponding early stage of COPD (2 weeks smoke exposure) were analyzed.FEV0.05, forced expiratory volume in 0.05 s.FEV0.05/FVC: ratio of forced expiratory volume in 0.05 s to forced vital capacity.(f ) Representative H&E images of lung tissues in the late stage of COPD (4 weeks smoke exposure) were shown.(g) The quantitative cell numbers calculated by flow cytometry between early stage and late stage of COPD were compared, among which γδ T cells and M2 macrophages showed a significant increase, so, in the following research, we focused on these two types of immune cells.(h) Representative confocal immunofluorescence images of lung tissue in the late stage of COPD.Blue color, DAPI; red color, TCR γδ; green color, CD206 or CD86.(i) In the late stage of COPD, we further observed periodontitis promoted the aggregation of IL 17 + γδ T cells and IFN γ + γδ T cells.Representative flow cytometry plots of IL 17 + γδ T cells and IFN γ + γδ T cells and corresponding quantitative analysis of lung tissue in late stage of COPD were shown.*P < 0.05, ns, not significant.B, blank control; P, periodontitis; C, COPD; CP, COPD with periodontitis.

9 FIG 5
FIG 5 Inhibition of γδ T cells affected periodontitis promoting COPD in vivo.(a) With or without the γδ-TCR monoclonal antibody treatment, the lung function test was performed in each group, and the results were shown as FEV0.05 value and FEV0.05/FVC value.FEV0.05, forced expiratory volume in 0.05 s.FEV0.05/FVC, ratio of forced expiratory volume in 0.05 s to forced vital capacity.(b) A representative H&E image of lung tissue in each group was shown.Periodontitis aggravated COPD, while γδ-TCR monoclonal antibody treatment partly reduced COPD severity.(c-g): Representative flow cytometry plots and corresponding quantitative analysis of lung tissue for each group were shown.(h) With or without γδ-TCR monoclonal antibody treatment, representative confocal immunofluorescence images of γδ T cells and M2 macrophages of lung tissue for each group were shown.Blue color, DAPI; red color, TCR γδ; green color, CD206.*P < 0.05, ns, not significant.B, blank control; P, periodontitis; C, COPD; CP, COPD with periodontitis; C-Anti, the COPD group with γδ-TCR monoclonal antibody treatment; CP-Anti, the CP group with γδ-TCR monoclonal antibody treatment.

FIG 6
FIG 6 Enhanced IFN γ and IL 17 levels and expression of M2-polarized genes in clinical COPD with periodontal pathogen BALF samples.(a) IFN γ and IL 17 levels in clinical COPD BALF samples were measured by ELISA.(b) Relative gene expression levels in clinical COPD BALF samples were quantified by RT-qPCR.*P < 0.05, ns, not significant.COPD-no P. g, BALF of COPD patients with no P. gingivalis detected; COPD-P.g, BALF of COPD patients with P. gingivalis detected.

FIG 7
FIG 7 The potential immune mechanism by which periodontitis aggravates COPD progression.Under periodontitis state, periodontitis-associated bacteria, as represented by P. gingivalis, migrate to and colonize on lung tissue and affect lung microbiota homeostasis.Following, periodontitis promotes the aggregation and activation of γδ T cells, with IL 17 and IFN γ directly promoting the progression of COPD.Simultaneously, IL 17 further induces M2 polarization of macrophages, thereby contributing to COPD progression.